P53 is the most commonly mutated tumor suppressor gene in human cancers. In the normal response to stress, it plays two main roles, causing either cell cycle arrest or apoptosis depending, in part, on the cell type. We have analyzed the posttranslational modifications to human p53 in response to three common genotoxic insults: ionizing radiation, which produces DNA double-strand breaks; UV light, which produces bulky DNA photoproducts and adriamycin, which is an anti-cancer agent and elicits a more complex response mediated in part through the ATR pathway. Our data revealed that Ser15, Ser20, and Thr18 are phosphorylation sites that are uniquely targeted by genotoxic agents but not by the non-genotoxic agents. Furthermore, we examined each of 14 p53 modification sites for site interdependence through the use of modification-specific antibodies. Strikingly, strong site-interdependencies were identified between three groups of sites: Ser6 and Ser9; Ser9, Ser15, Thr18, and Ser20; and Ser33 and Ser37. For example, changing Ser15 to alanine completely abrogated phosphorylation of p53 at Ser9, Ser20 and Thr18, while changing Ser20 to alanine only abrogated phosphorylation at Thr18. These data have allowed us to propose a new mechanism that may serve to prevent inappropriate or premature activation of p53. This mechanism complements our previously described mechanism of enzyme activation cascades. Phosphorylation of mouse p53 at Ser18 occurs after DNA damage. To determine the physiological roles of this phosphorylation in p53-dependent DNA damage responses, we introduced a Ser18 to Ala missense mutation into the endogenous p53 gene. Although the mice were not susceptible to spontaneous tumorigenesis, thymocytes and mouse embryonic fibroblasts (MEF) showed abnormal p53-dependent DNA damage responses. Analysis of p53 responses to ionizing radiation (IR) in p53 mutant thymocytes indicated that this phosphorylation is not required for p53 stabilization, but influences expression of p53-dependent genes in a promoter-specific manner and diminishes p53-dependent apoptosis. In p53 mutant MEFs, p53 was more stable than in wild-type MEFs after UV irradiation, whereas p53-dependent functions and p53 acetylation were impaired. Chromatin immunoprecipitation analysis showed normal in vivo binding of p53 mutant to p53-dependent promoters, suggesting that neither Ser18 phosphorylation nor acetylation is required for binding of p53 to p53-dependent promoters in these cells following DNA damage. These findings provide novel insights into the role of murine p53 Ser18 phosphorylation in p53-dependent responses to genotoxic stresses and have important implications for future functional studies of p53 posttranslational modifications. Wip1 phosphatase (PPMD1) is a member of the PP2C family of evolutionary conserved protein phosphatases. Originally described as a p53-regulated gene, Wip1 subsequently was implicated as a negative regulator of p53 functions through its ability to attenuate p38 MAPK activity, thereby mediating the inactivation of p53 in response to different cellular stresses. The importance of this negative-feedback loop was further illustrated by the findings that Wip1 phosphatase complements several oncogenes for cell transformation in vitro and that PPM1D is amplified and overexpressed in human primary breast cancers, neuroblastomas and ovarian clear cells adenocarcinomas. Taking advantage of our recently generated knockout mice with a disrupted Wip1 gene, we have characterized the ability of Wip1-null mouse embryo fibroblasts (MEFs) to undergo transformation in vitro, as well as in vivo, by crossing breast cancer prone mouse strains into a Wip1-null background. Our results show that inactivation of the Wip1 phosphatase gene, Ppm1d, rendered cells resistant to tumorigenic transformation by several oncogenes both in vitro and in vivo. A detailed analysis of Wip1-null cells revealed that deregulation of the cyclin D1-Cdk4-pRb pathway due to constitutive induction of the p16 Ink4a protein could be a major mechanism for this phenomenon. The modulation of p16 Ink4a levels through inactivation or depletion of the Wip1 phosphatase may be an effective new approach to the treatment of human primary breast tumors.